Parvalbumin in Human Brain

Parvalbumin was isolated from human cerebral cortex and biceps and triceps muscles by HPLC. The immunological properties of the human protein and the mobility in two-dimensional polyacrylamide gels were similar to that of parvalbumin isolated from the muscles of rat, mouse, rabbit, and chicken. The tryptic peptide maps of the human parvalbumin, however, differed considerably from all other parvalbumins, indicating a distinct primary structure. The immunolabeled cells in the hippocampus of the human brain were of different sizes and forms; they occurred in all subfields and probably represent interneurons.     

Parvalbumin, a Neuronal Protein in Brain Cell Cultures

Dissociated brain cell cultures were derived from 14-day-old embryonic as well as from newborn mice. The cells were grown in a medium containing 10% fetal calf serum. Indirect immunofluorescence was performed using antisera directed against the Ca2+-binding protein parvalbumin (Mr 12,000). In embryonic cultures a large proportion of cells was intensely stained by antiparvalbumin . In double-labelling experiments involving the simultaneous application of antisera against parvalbumin and the neuron-specific enolase, the enolase-containing cells were also parvalbumin-positive and both antisera revealed identical intracellular staining patterns. Conversely, almost no parvalbumin- and enolase-positive cells were present in cultures derived from newborn mice. However, in these cultures many cells were immunoreactive toward the myelin basic protein, an accepted marker for oligodendrocytes. The presence of parvalbumin within the embryonic brain cell cultures was confirmed by analyses of the culture extracts (4 mM EDTA, pH 7.5) by HPLC on reverse-phase supports, two-dimensional polyacrylamide gel electrophoresis, and immunoblotting. The present study suggests that in mouse brain cell cultures, parvalbumin is localized in neurons.     

Developmental and Functional Studies of Parvalbumin and Calbindin D28K in Hypothalamic Neurons Grown in Serum-Free Medium

The Ca2+-binding proteins parvalbumin (Mr = 12K) and calbindin D28K [previously designated vitamin D-dependent Ca2+-binding protein (Mr = 28K)] are neuronal markers, but their functional roles in mammalian brain are unknown. The expression of these two proteins was studied by immunocytochemical methods in serum-free cultures of hypothalamic cells from 16-day-old fetal mice. Parvalbumin is first detected in all immature neurons, but during differentiation, the number of parvalbumin-immunoreactive neurons greatly declines to a level reminiscent of that observed in vivo, where only a subpopulation of neurons stains for parvalbumin. In contrast, calbindin D28K was expressed throughout the period investigated only in a distinct subpopulation of neurons. Depolarization of fully differentiated hypothalamic neurons in culture resulted in a dramatic decrease of parvalbumin immunoreactivity but not of calbindin D28K immunoreactivity. The parvalbumin staining was restored on repolarization. Because the anti-parvalbumin serum seems to recognize only the metal-bound form of parvalbumin, the loss of immunoreactivity may signal a release of Ca2+ from intracellular parvalbumin during depolarization of the cells. We suggest that parvalbumin might be involved in Ca2+-dependent processes associated with neurotransmitter release.     

Rat Brain S100b Protein: Purification, Characterization, and Ion Binding Properties. A Comparison with Bovine S100b Protein

We purified to homogeneity rat brain S100b protein, which constitutes about 90% of the soluble S100 protein fraction. Purified rat S100b protein comigrates with bovine S100b protein in nondenaturant system electrophoresis but differs in its amino acid composition and in its electrophoretic mobility in urea-sodium dodecyl sulfate-polyacrylamide gel with bovine S100b protein. The properties of the Ca2+ and Zn2+ binding sites on rat S100b protein were investigated by flow dialysis and by fluorometric titration, and the conformation of rat S100b in its metal-free form as well as in the presence of Ca2+ or Zn2+ was studied. The results were compared with those obtained for the bovine S100b protein. In the absence of KCl, rat brain S100b protein is characterized by two high-affinity Ca2+ binding sites with a KD of 2 X 10(-5) M and four lower affinity sites with KD about 10(-4) M. The calcium binding properties of rat S100b protein differ from bovine S100b only by the number of low-affinity calcium binding sites whereas similar Ca2+-induced conformational changes were observed for both proteins. In the presence of 120 mM KCl rat brain S100b protein bound two Zn2+-ions/mol of protein with a KD of 10(-7) M and four other with lower affinity (KD approximately equal to 10(-6) M). The occupancy of the two high-affinity Zn2+ binding sites was responsible for most of the Zn2+-induced conformational changes in the rat S100b protein. No increase in the tyrosine fluorescence quantum yield after Zn2+ binding to rat S100b was observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structures of EF-hand Ca 2+-binding proteins: Diversity in the organization, packing and response to Ca 2+ Binding

The growing database of three-dimensional structures of EF-hand calcium-binding proteins is revealing a previously unrecognized variability in the coformations and organizations of EF-hand binding motifs. The structures of twelve different EF-hand proteins for which coordinates are publicly available are discussed and related to their respective biological and biophysical properties. The classical picture of calcium sensors and calcium signal modulators is presented, along with variants on the basic theme and new structural paradigms.© Kluwer Academic Publishers     

Isolation of a Ca2+-Dependent Actin-Fragmenting Protein from Brain, Spinal Cord, and Cultured Neurones

Extracts of ox spinal cord and chicken brain were fractionated by ion-exchange chromatography and assayed for their ability to reduce the viscosity of muscle F-actin solutions. Two distinct peaks of activity were obtained, one of which was further purified by affinity chromatography on a DNAase-actin Sepharose column. Following molecular exclusion chromatography, the actin component appeared as a complex of 1 molecule of a protein with molecular weight 90,000 and 2 molecules of actin (42,000). This tightly bound complex was resistant to most methods of protein separation, but was resolvable into its component proteins by sodium dodecyl sulphate acrylamide gel electrophoresis. The protein of molecular weight 90,000 could be eluted from such a gel in a fully active form. The activity of the protein from ox spinal cord was closely similar to that of gelsolin, an actin-fragmenting protein originally isolated from rabbit lung macrophages. Like gelsolin, the protein from ox spinal cord produced fragmentation of muscle F-actin filaments at Ca2+ concentrations greater than 10(-7) M, and had a nucleating effect on the polymerisation of muscle actin; the latter was measured most easily by the enhancement of fluorescence of muscle actin conjugated to N-(1-pyrenyl)iodoacetamide. Nucleation was more effective in the presence of Ca2+, but also occurred in its absence, and the same was true of complex formation between the 90,000 protein and muscle G-actin. On the basis of its actin-fragmenting activity, we estimate that the 90,000 molecular weight protein constitutes 0.2% of the protein initially extracted from ox spinal cord. A very similar protein, indistinguishable in its action on actin but containing variable amounts of a protein of molecular weight 85,000 as well as 90,000, was isolated from chicken brain. A similar protein was also detected in pure cultures of sympathetic neurones by enrichment on a DNAase-actin affinity column and by immune blotting and by immunofluorescence. We conclude that a protein similar, if not identical to macrophage gelsolin is present in neurones and that it probably plays a part in the actin-based movements of these cells.     

Isolation, Characterization, and Synthesis of AlphaFetoprotein from Neonatal Rat Brain

Monospecific anti-rat serum alpha-fetoprotein (AFP) IgG was coupled to cyanogen bromide-activated Sepharose-4B (4.5 mg/ml packed volume of gel) to yield an immunoaffinity matrix. The immunoaffinity column was used to isolate AFP from feto-neonatal rat brain. The purified AFP was immunologically and electrophoretically similar to serum AFP. It yielded a single band with a molecular weight of 70,000 on sodium dodecyl sulphate polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis of the protein under nondenaturing conditions yielded two charge variants of AFP, reminiscent of AFP from feto-neonatal rat serum. The AFP was observed to bind estradiol with Ka = 5.8 X 10(8) M -1 and 1.3 X 10(8) M -1 by dextran-coated charcoal adsorption and Sephadex gel filtration techniques, respectively. Newborn rat brain cells linearly incorporated [14C]leucine into immunoprecipitable AFP during 6 h in culture. It is, therefore, concluded that feto-neonatal rat brain contains AFP similar to that present in fetal serum and that it may arise in brain as a result of its in situ synthesis.     

New perspectives on S100 proteins: a multi-functional Ca 2+ -, Zn 2+ - and Cu 2+ -binding protein family

S100 proteins (16 members) show a very divergent pattern of cell- and tissue-specific expression, of subcel-lular localizations and relocations, of post-translational modifications, and of affinities for Ca 2+ , Zn 2+ , and Cu 2+ , consistent with their pleiotropic intra- and extracellular functions. Up to 40 target proteins are reported to interact with S100 proteins and for S100A1 alone 15 target proteins are presently known. Therefore it is not surprising that many functional roles have been proposed and that several human disorders such as cancer, neurodegenerative diseases, cardiomyopathies, inflammations, diabetes, and allergies are associated with an altered expression of S100 proteins. It is not unlikely that their biological activity in some cases is regulated by Zn 2+ and Cu 2+ , rather than by Ca 2+ Despite the numerous putative functions of S100 proteins, their three-dimensional structures of, e.g., S100B, S100A6, and S100A7 are surprisingly similar. They contain a compact dimerization domain whose conformation is rather insensitive to Ca 2+ binding and two lateral a-helices III and III, which project outward of each subunit when Ca 2+ is bound. Target docking depends on the two hydrophobic patches in front of the paired EF-hand generated by the binding of Ca 2+. The selec-tivity in target binding is assured by the central linker between the two EF-hands and the C-terminal tail. It appears that the S100-binding domain in some target proteins contains a basic amphiphilic a-helix and that the mode of interaction and activation bears structural similarity to that of calmodulin.© Kluwer Academic Publishers     

Biochemical Characterization of Annexins I and II Isolated from Pig Nervous Tissue

Five proteins having molecular masses of 90, 67, 37, 36, and 32 kDa (p90, p67, p37, p36, and p32, respectively) were identified in the particulate fractions of pig brain cortex and pig spinal cord prepared in the presence of 0.2 mM Ca2+ and further purified using a protocol previously described for the purification of calpactins. Proteins p90, p37, and p36 are related to annexins I and II. Annexin II, represented by p90, is found as an heterotetramer, composed of two heavy chains of 36 kDa and two light chains of 11 kDa, and as a monomer of 36 kDa. Protein p37, which differs immunologically from p36, is a monomer and could be related to annexin I. All three proteins are Ca(2+)-dependent phospholipid- and F-actin-binding proteins; they are phosphorylated on a serine and on a tyrosine residue by protein kinases associated with synaptic plasma membranes. Purified p36 monomer and p36 heterotetramer proteins bind to actin at millimolar Ca2+ concentrations. The stoichiometry of p36 binding to F-actin at saturation is 1:2, corresponding to one tetramer or monomer of calpactin for two actin monomers (KD, 3 x 10(-6) M). Synaptic plasma membranes supplemented with the monomeric or tetrameric forms of p36 phosphorylate the proteins on a serine residue. The monomer is phosphorylated on a serine residue by a Ca(2+)-independent protein kinase, whereas the heterotetramer is phosphorylated on a serine residue and a tyrosine residue by Ca(2+)-dependent protein kinases. Antibodies to brain p37 and p36 together with antibodies to lymphocytes lipocortins 1 and 2 were used to follow the distribution of these proteins in nervous tissues. Polypeptides of 37, 34, and 36 kDa cross-react with these antibodies. Anti-p37 and antilipocortin 1 cross-react on the same 37- and 34-kDa polypeptides; anti-p36 and antilipocortin 2 cross-react only on the 36-kDa polypeptides.     

An interaction-based analysis of calcium-induced conformational changes in Ca2+ sensor proteins.

Calcium sensor proteins translate transient increases in intracellular calcium levels into metabolic or mechanical responses, by undergoing dramatic conformational changes upon Ca2+ binding. A detailed analysis of the calcium binding-induced conformational changes in the representative calcium sensors calmodulin (CaM) and troponin C was performed to obtain insights into the underlying molecular basis for their response to the binding of calcium. Distance difference matrices, analysis of interresidue contacts, comparisons of interhelical angles, and inspection of structures using molecular graphics were used to make unbiased comparisons of the various structures. The calcium-induced conformational changes in these proteins are dominated by reorganization of the packing of the four helices within each domain. Comparison of the closed and open conformations confirms that calcium binding causes opening within each of the EF-hands. A secondary analysis of the conformation of the C-terminal domain of CaM (CaM-C) clearly shows that CaM-C occupies a closed conformation in the absence of calcium that is distinct from the semi-open conformation observed in the C-terminal EF-hand domains of myosin light chains. These studies provide insight into the structural basis for these changes and into the differential response to calcium binding of various members of the EF-hand calcium-binding protein family. Factors contributing to the stability of the Ca2+-loaded open conformation are discussed, including a new hypothesis that critical hydrophobic interactions stabilize the open conformation in Ca2+ sensors, but are absent in "non-sensor" proteins that remain closed upon Ca2+ binding. A role for methionine residues in stabilizing the open conformation is also proposed.     

Molecular Cloning of Two Additional Members of the Neural Visinin-Like Ca2+-Binding Protein Gene Family

Abstract: We have isolated a rat cDNA clone encoding a neural visinin-like Ca²⁺-binding protein (NVP), which we designate NVP-1. To identify additional molecular forms of NVP, a rat brain cDNA library was screened for their presence using an NVP-1 cDNA probe under low-stringency hybridization conditions. Two types of cDNA clones encoding structurally related proteins, designated NVP-2 and NVP-3, have been isolated. The deduced amino acid sequences of NVP-2 and NVP-3 are 89.0% and 68.6% identical to that of NVP-1, respectively, and contain consensus sequences for EF-hand Ca²⁺-binding sites. Northern blot analysis shows that NVP-1, NVP-2, and NVP-3 mRNAs are most highly expressed in brain and are differentially expressed in various regions of rat brain. These results suggest that NVP-2 and NVP-3 are additional members of the NVP gene family.     

Possible mechanism for the influence of weak magnetic fields on biological systems

A physical mechanism is suggested for a resonant interaction of weak magnetic fields with biological systems. An ion inside a Ca(2+)-binding protein is approximated by a charged oscillator. A shift in the probability of ion transition between different vibrational energy levels occurs when a combination of static and alternating magnetic fields is applied. This in turn affects the interaction of the ion with the surrounding ligands. The effect reaches its maximum when the frequency of the alternating field is equal to the cyclotron frequency of this ion or to some of its harmonics or sub-harmonics. A resonant response of the biosystem to the magnetic field results. The proposed theory permits a quantitative explanation for the main characteristics of experimentally observed effects.     

Detection and Characterization of Some New Basic Proteins in Chicken Postsynaptic Densities

Chicken brain postsynaptic density (PSD) polypeptides, obtained by treating synaptosomes with 0.5% Triton X-100 and then further purified on a sucrose gradient, are demonstrated to contain four basic proteins of 76K (pI greater than 9.2), 58K (pI 8.1-8.8, heterogeneous), 40K (pI 9.0), and 24K (pI 8.9). Nonequilibrium pH gradient-sodium dodecyl sulfate two-dimensional gels further reveal six more basic proteins with pI values higher than 9.2: 76K, 52K, 47K, 45K, 36K, and 34K. These basic proteins are a major part of the total chicken PSD polypeptides appearing on the gels. Some of these basic proteins (58K, 52K, 47K, 36K, 24K, and two at 76K) are distinguishable from those of brain mitochondria, the major contaminant. The 40K and 34K proteins may be common mitochondrial polypeptides. The 45K protein is probably a mitochondrial contaminant. A number of proteins including 76K (synapsin I-like protein) and 58K, along with some other minor ones, can be phosphorylated by endogenous protein kinase(s) in the presence of Ca2+, Mg2+, and [gamma-32P]ATP. No PSD basic proteins bind Ca2+.     

Criticism of Lednev's mechanism for the influence of weak magnetic fields on biological systems.

V. V. Lednev has proposed a mechanism that he suggests would allow very weak magnetic fields, at the cyclotron resonance frequency for Ca2+ ions in the earth's field, to induce biological effects. I show that for four independent reasons no such mechanism can operate.     

Kinetic Characterization of Ca2+ Transport in Synaptic Membranes

Lysed synaptosomal membranes were prepared from brain cortices of HA/ICR Swiss mice, and the ATP-stimulated Ca2+ uptake, Ca2+-stimulated Mg2+-dependent ATPase activity, and the Ca2+-stimulated acyl phosphorylation of these membranes were studied. The Km values for free calcium concentrations ([Ca2+]f) for these processes were 0.50 microM, 0.40 microM, and 0.31 microM, respectively. Two kinetically distinct binding sites for ATP were observed for the ATP-stimulated Ca2+ uptake and the Ca2+-stimulated Mg2+-ATPase activity. The high-affinity Km values for ATP for these two processes were 16.3 microM and 28 microM, respectively. These results indicate that the processes studied operate in similar physiological concentration ranges for the substrates [Ca2+]f and ATP under identical assay conditions and, further, that these processes may be functionally coupled in the membrane.     

Identification and Characterization of Calcium-Dependent Metalloproteases in Rat Brain

We have begun to identify and characterize brain protease activities separated by and assayed in substrate-containing polyacrylamide gels. In the present report, we focus on four proteolytic activities identified from rat brain that are dependent on micromolar and millimolar Ca2+ concentrations for activity. In contrast to the previously described Ca2+-dependent neutral cysteine proteases (calpains), all four activities appear to be metalloproteases based on their inhibition by EDTA, EGTA, and 1,10-o-phenanthroline, but not by blockers of serine, cysteine, or aspartic proteases. In the presence of excess Ca2+ and the Zn2+-chelating inhibitor 1,10-o-phenanthroline, activity of the enzymes was reconstituted by addition of lower concentrations of Zn2+, and inhibited by higher Zn2+ concentrations. The four metalloproteases were designated MP-112, MP-92, MP-70, and MP-65 on the basis of their apparent molecular masses in kilodaltons. MP-70, the major activity detected, had an apparent kact for Ca2+ greater than 100 microM versus 10-25 microM for MP-65 and 50-100 microM for MP-92. MP-112 was a minor activity for which Ca2+ activation levels were not determined. MP-112, MP-70, and MP-65 were similar in being most active in the soluble fraction of 7-day neonate forebrain. In contrast, MP-92 activity was highest in the particulate fraction of adult forebrain. About half of the MP-92 activity and lower levels of the other three activities were still detectable in particulate fractions after detergent extraction of membrane, suggesting an association with cytoskeletal or other structural proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Somatostatin Binding Sites in Human and Monkey Brain: Localization and Characterization

A radioiodinated analogue of somatostatin 28, 125I [Leu8,D-Trp22,Tyr25] SS-28, was used to localize and characterize somatostatin binding sites in both human and monkey brain. High-affinity binding sites (approximately 1 nM) were found in cerebral cortex. The highest binding was in cerebral cortex with intermediate binding found in hippocampus, striatum, and amygdala and low binding in hypothalamus and brainstem. There was a rough correlation between somatostatin receptor binding and concentrations of somatostatin-like immunoreactivity (SLI) in human brain. Somatostatin receptors were stable for up to 24 h in an animal model simulating human autopsy conditions and there was no correlation between postmortem interval and receptor binding in human brain. Pharmacologic characterization in human cortex showed that there was a correlation between the inhibition of receptor binding by somatostatin analogues and their known abilities to inhibit growth hormone secretion. These findings demonstrate that a highly specific membrane-associated receptor for somatostatin is present in both monkey and human brain. Examination of somatostatin receptor binding in Alzheimer's disease and Huntington's disease may improve understanding of the role of somatostatin in both these illnesses.     

P100, a transcriptional coactivator,is a human homologue of staphylococcal nuclease.

Staphylococcus aureus nuclease (SNase) homologues, previously thought to be restricted to bacteria and archaea, are demonstrated by sequence analysis to be present also in eukaryotes. The human cellular coactivator p100 is shown to contain four repeats, each of which is a SNase homologue. Surprisingly, these repeats are unlikely to possess SNase-like activities as each lacks equivalent SNase catalytic residues, yet they may mediate p100''s single-stranded DNA-binding function. Products of Corydalis sempervirens and Saccharomyces cerevisiae open reading frames are predicted to adopt the same fold and possess similar functions as SNase. Five additional hypothetical proteins of bacterial origin are also predicted to be active SNase-like nucleases, including one that appears to be C-terminally truncated in a manner analogous to an engineered active SNase variant. Conservation of Asp-19 and Asp-83 among these homologues suggests a re-evaluation of the roles of these residues in Ca(2+)-binding and/or catalysis.